microRNA调控肝细胞内源性免疫信号网络抗HBV的特征分析及白花香莲解毒方干预机制研究

There is a enormous difference in the chronicity rate after HBV infection in different ages, which indicate that the key factor on HBV infection prognosis is the intrinsic immune mechanisms of inhibiting/clear of HBV. But which immune signaling pathways involved in anti-HBV process in hepatocytes, as well as how to regulate is currently still poorly understood. Based on our preliminary study, we propose that microRNA is endogenous regulator of immune signaling network in liver cells, play a key role in the anti-HBV mechanism, Baihua xianglian detoxification formula is antagonist HBV through the intervention of microRNA mechanism. This study applied microRNA chips, qRT-PCR, etc., to obtain endogenous hepatocytes differentially expressed microRNA map in self-healing and chronicity patients, followed by bioinformatics analysis to build anti-HBV microRNA-immune signaling regulatory network, then verify and clarify microRNA and immune signaling pathways（positive regulation, negative regulation）with the most anti-HBV activity, as well as the molecular mechanism of synergistically inhibit/clear HBV via microRNA antisense oligonucleotide interference technology, to illuminate the anti-HBV mechanism of Baihua xianglian detoxification formula with the function of detoxification supplement deficiency.

不同年龄层次的人群感染HBV后，发生慢性化的机率存在着巨大的差异性。这提示机体存在抑制/清除HBV的内在免疫机制，是决定HBV感染预后的关键。但目前对宿主（肝细胞）内有哪些免疫信号通路参与抗HBV过程，通过何种途径调控，尚知之甚少。基于前期研究，我们提出了"microRNA是肝细胞内源性免疫信号网络的调控子，在抗HBV机制上发挥关键性作用，白花香莲解毒方可通过干预该机制拮抗HBV"的假说，并首先应用microRNA芯片、qRT-PCR等绘制HBV感染自愈者及慢性化者肝细胞内差异性表达的microRNA图谱，继而通过生物信息学构建抗HBV的microRNA-免疫信号调控网络，最后应用microRNA反义寡核苷酸干扰等技术鉴定与明确肝细胞内最具抗HBV活性的microRNA、免疫信号通路（正调控、负调控）及彼此协同抑制/清除HBV的分子机理，并揭示壮药白花香莲解毒方"解毒补虚"抗HBV的机制。

本实验采用miRNA芯片、基因芯片及生物信息学等多种研究手段，筛选及验证肝细胞内抗HBV的miRNA、mRNA表达谱，揭示壮药白花香莲解毒方抑制HBV复制与表达的疗效机制，并为寻找新的抗HBV治疗靶点提供理论基础。结果发现（1）新构建的HBV1.3P-HepG2细胞模型能表达HBV DNA、cccDNA、HBsAg、HBeAg等HBV标志物。（2）白花香莲解毒方在0.625mg干膏/ml浓度、干预48h时对HBV DNA复制的抑制率达41.85±2.35%。（3）miRNA芯片筛选出20个与白花香莲解毒方抑制HBV相关的miRNA。mRNA芯片筛选出183个与白花香莲解毒方抑制HBV相关的mRNA。通过生物信息学筛选出miR141-3P/PPARα、miR372-3p/NFIB、miR20a-5p/IL-8、miR-106a-5p/IL-8共4组miRNA/mRNA进行抗HBV的功能验证。（4）在2种细胞模型中，miR-372-3p能够促进HBV DNA复制及HBsAg表达，其HBV DNA水平分别是空白对照组的1.37倍、1.36倍（P＜0.05）。miR-141-3p能抑制HBV DNA复制及HBsAg表达，其HBV DNA水平分别是空白对照组的59%、64%（P＜0.05）。miR-20a-5p的HBV DNA水平分别是空白对照组的96%、1.03倍（P＞0.05）。miR-106a-5p的HBV DNA水平分别是空白对照组的94%、95%（P＞0.05）。（5）在2种细胞模型中，PPARα能促进HBV DNA复制及HBsAg表达，其HBV DNA水平分别是空白对照组的1.46倍、1.27倍（P＜0.05）。NFIB能抑制HBV DNA复制及HBsAg表达，其HBV DNA水平分别是空白对照组的76%、55%（P＜0.05）。IL-8能促进HBV DNA复制及HBsAg表达，其HBV DNA水平分别是空白对照组的1.54倍、1.62倍（P＜0.05）。（6）miRNA/mRNA共转染抗HBV的功能验证显示，miR-141-3p和PPARα共转染的HBV DNA水平分别是只转入PPARα的78%、85%（P＜0.05）。miR-372-3p和NFIB共转染的HBV DNA水平分别是只转入NFIB的1.52倍、1.75倍（P＜0.05）。